The sole mediators of exchange between the nuclear and cytoplasmic compartments are nuclear pore complexes (NPCs), comprised of proteins termed nucleoporins or Nups. Nucleocytoplasmic transport is driven by soluble transport factors (most belonging to a related family of proteins termed karyopherins or Kaps) that carry their cognate cargos across the NPC. This transport is regulated at multiple levels, including cargo recognition by karyopherins and interactions with the NPC. The NPC also plays a key regulatory role in gene expression by influencing nuclear architecture and acting as a point of control for various nuclear processes. Major pathological cellular processes are associated with altered nucleocytoplasmic transport, and many viruses target components of the nucleocytoplasmic transport pathway to usurp it. Hence, nucleoporins and transport factors are key potential targets for drug therapy. We will focus on two of the key regulatory areas of nucleocytoplasmic transport, where structural information is most likely to lead to fundamental mechanistic insights into these regulatory processes. First, we will investigate how Kaps recognize their cargos. The overlapping specificity of Kaps endows cells with the ability to selectively control the transport of thousands of cargos and provides a rich source of potential targets for pharmacological intervention. PSI Biology will provide crystal structures of Kaps bound to their cargos, which will complement biochemical and bioinformatics approaches to reveal both the sequence and structure requirements for cargo recognition. Second, we will map at high resolution the basket region of the NPC, a critical point of control for a bewildering array of nuclear processes. These processes are mediated by the interplay of interactions among the basket proteins;however, assigning these varied functions to domains of the basket proteins has proven largely unsuccessful, primarily because little is known about its structural organization and the interdependence of its components. The atomic structures of basket components and their interactors from PSI Biology will therefore be integrated with our complementary biophysical, morphological, and proteomic data on selected subcomplexes associated with the NPC to obtain a high resolution map of the nuclear basket in the context of the NPC and nuclear periphery. Once established, this map will be used to guide the dissection and perturbation of individual components, to elucidate the relationship between the structural organization of the basket and the mechanisms by which it executes its various functions. PUBLIC HEALTH RELEVANCE: Major pathological cellular processes are associated with malign alterations in the pathways that transport materials to and from the cell's nucleus. This project promises to characterize key areas of the nuclear transport pathways at the atomic scale, to discover potential drug targets. This will ultimately open the door to new therapies to control nuclear transport, either to thwart viruses that are usurping these pathways or to correct defects in these pathways that result in developmental or oncogenic diseases.